Paroxysmal Cold Hemoglobinuria’ Case Report with Characterization of the Donath -Landsteiner Hemobsin SHARON FISHER, M.D. Tel-Aviv, Israd A case of nonsyphilitic paroxysmal cold hemoglobinuria is presented in which hemolytic attacks recurred ten years after splenectomy. Immunosuppressive treat- ment (Meticorten,@ azathioprine) was of questionable benefit. The Donath-Land- Steiner hemolysin in this case was isolated by elution from red cells and by DEAE cellulose chromatography of the patient’s serum and characterized as a type L IgG globulin. Certain complement components were found to be required in the cold phase, others in the warm phase of the Donath-Landsteiner reaction. The positive antiglobulin test in paroxysmal cold hemoglobinuria is complement and temperature dependent. P AROXYSMAL cold hemoglobinuria is a rare autoimmune hemolytic anemia caused by the circulating hemolysins first described by Donath and Landsteiner [I]. The clinical pic- ture of this disease is well known and its sero- logic characteristics have been extensively studied and thoroughly reviewed [2]. Still un- der discussion are the problems of specific ther- apy and that of the immunochemical charac- teristics of the Donath-Landsteiner antibodies. The present report concerns a case of non- syphylitic paroxysmal cold hemoglobinuria with interesting clinical data, in which certain molecular properties of the Donath-Land- Steiner hemolysin were studied and defined. CASE REPORT This nineteen year old girl of Jewish-Sephardic extraction (NM.) was referred to our hematologic clinic in May 1965 for evaluation and follow-up. Her disease, a chronic hemolytic anemia with marked splenomegaly, was first diagnosed in 1956, at the age of six, when her blood picture was as follows: hemoglobin, 6 gm. per cent: erythrocytes, 2,000,OOO per cu. mm.; reticulocytes, 30 pro mil; serum bilirubin, 1.6 mg. per cent, almost all indi- rect: serum iron, 85 gamma per cent. The bone marrow was hypercellular with marked erythro- blastic hyperplasia. In 1959 the patient was sub- jected to a splenectomy, followed by apparent re- covery. The spleen weighed 1,500 gm. and on path- ologic examination showed severe congestion with marked atrophy of the lymphatic tissue and large deposits of hemosiderin in the sinusoids. When first examined in our clinic the girl had no com- plaints, the physical examination was within nor- mal limits and the blood picture was apparently normal, although the hemoglobin and hematocrit levels were lower and the reticulocyte counts higher during the colder months of the year (Fig. 1). Com- plementary examination showed normal hemoglo- bin electrophoresis; hemoglobin F, 0.6 per cent; Coombs’ test, negative; glucose-6-phosphate dehy- drogenase, 10 units per ml. blood; and a 10 per cent eosinophilia in the peripheral blood. In Fcb- ruary 1967 the patient suddenly became severely ill with fever, chills, pallor and dark urine. She had attacks of severe pain in the lower extremities especially in the toes and the metatarsal region, with discoloration of the skin in these areas, typi- cal of Raynaud’s phenomenon. The pains and vaso- motor disturbances disappeared after warming of the affected areas. The blood picture was one of severe hemolytic anemia: hemoglobin, 6.7 gm. per cent: hematocrit, 20 per cent; reticulocytes, 130 pro mil; leuko- cytes, 8,000 per cu. mm.; platelets, 300,000 per cu. mm. Protein electrophoresis was normal and no cryoglobulins were found. The cold agglutinins and the Ham tests were * From the Department of Hematology, Jaffa Government Hospital, Tel-Aviv, Israel. Manuscript rchceivetl April 4, 1968. “01. 46, MARCUS 1969 475
Transcript
Paroxysmal Cold Hemoglobinuria’
Case Report with Characterization of the Donath -Landsteiner Hemobsin
SHARON FISHER, M.D.
Tel-Aviv, Israd
A case of nonsyphilitic paroxysmal cold hemoglobinuria is presented in which hemolytic attacks recurred ten years after splenectomy. Immunosuppressive treat- ment (Meticorten,@ azathioprine) was of questionable benefit. The Donath-Land- Steiner hemolysin in this case was isolated by elution from red cells and by DEAE cellulose chromatography of the patient’s serum and characterized as a type L IgG globulin. Certain complement components were found to be required in the cold phase, others in the warm phase of the Donath-Landsteiner reaction. The positive antiglobulin test in paroxysmal cold hemoglobinuria is complement and temperature dependent.
P AROXYSMAL cold hemoglobinuria is a rare autoimmune hemolytic anemia caused by
the circulating hemolysins first described by Donath and Landsteiner [I]. The clinical pic- ture of this disease is well known and its sero- logic characteristics have been extensively studied and thoroughly reviewed [2]. Still un- der discussion are the problems of specific ther- apy and that of the immunochemical charac- teristics of the Donath-Landsteiner antibodies. The present report concerns a case of non- syphylitic paroxysmal cold hemoglobinuria with interesting clinical data, in which certain molecular properties of the Donath-Land- Steiner hemolysin were studied and defined.
CASE REPORT
This nineteen year old girl of Jewish-Sephardic extraction (NM.) was referred to our hematologic clinic in May 1965 for evaluation and follow-up. Her disease, a chronic hemolytic anemia with marked splenomegaly, was first diagnosed in 1956, at the age of six, when her blood picture was as follows: hemoglobin, 6 gm. per cent: erythrocytes, 2,000,OOO per cu. mm.; reticulocytes, 30 pro mil; serum bilirubin, 1.6 mg. per cent, almost all indi- rect: serum iron, 85 gamma per cent. The bone marrow was hypercellular with marked erythro- blastic hyperplasia. In 1959 the patient was sub-
jected to a splenectomy, followed by apparent re- covery. The spleen weighed 1,500 gm. and on path- ologic examination showed severe congestion with marked atrophy of the lymphatic tissue and large deposits of hemosiderin in the sinusoids. When first examined in our clinic the girl had no com- plaints, the physical examination was within nor- mal limits and the blood picture was apparently normal, although the hemoglobin and hematocrit levels were lower and the reticulocyte counts higher during the colder months of the year (Fig. 1). Com- plementary examination showed normal hemoglo- bin electrophoresis; hemoglobin F, 0.6 per cent; Coombs’ test, negative; glucose-6-phosphate dehy- drogenase, 10 units per ml. blood; and a 10 per cent eosinophilia in the peripheral blood. In Fcb- ruary 1967 the patient suddenly became severely ill with fever, chills, pallor and dark urine. She had attacks of severe pain in the lower extremities especially in the toes and the metatarsal region, with discoloration of the skin in these areas, typi- cal of Raynaud’s phenomenon. The pains and vaso- motor disturbances disappeared after warming of the affected areas.
The blood picture was one of severe hemolytic anemia: hemoglobin, 6.7 gm. per cent: hematocrit, 20 per cent; reticulocytes, 130 pro mil; leuko- cytes, 8,000 per cu. mm.; platelets, 300,000 per cu. mm. Protein electrophoresis was normal and no cryoglobulins were found.
The cold agglutinins and the Ham tests were
* From the Department of Hematology, Jaffa Government Hospital, Tel-Aviv, Israel. Manuscript rchceivetl April 4, 1968.
“01. 46, MARCUS 1969 475
476 Paroxysmal Cold Hemoglobinuria-Fisher
40
*
B 20 ;
I I
1965 1966 1967
FIG. 1. Hemoglobin, hematocrit and reticulocytes, 1965-1967.
negative, but the Donath-Landsteiner test was posi- tive. The Kahn, Wassermann and Nelson tests were negative.
The patient was kept in a warm environment; treatment with Meticorten was begun and one week later an improvement in the blood picture was noted. During the following weeks the weather became warm and the clinical picture improved continuously even after steroid treatment was dis- continued (Fig. 1). At the beginning of the winter 1967-1968 the patient was given prophylactically
TABLE I
THE REQUIREMENTS OF COMPLEMENT IN THE DONATH-
LANDSTEINER REACTION
Tube No.
Data 1234567
Incubation thirty min. at 2’c. of
Normal red cells (0 RhS) +++++++
Patient’s serum ++++ Normal serum (or
guinea pig serum) ++++ +
Patient’s serum, inactivated at 56 Oc. + +
Washing of cells with cold sodium chloride (0.85 per cent) No Yes Yes Yes No No No
Incubation 60 min. at 37°C. afler addition of
Normal serum + + Normal serum in-
activated + Sodium chloride,
0.15 M +
Hemolysis 3 3 11000
a course of Meticorten, 1 mg. per kg. weight per day for six weeks, and subsequently azathioprine, 100 mg. per day. Administration of the latter drug had to be discontinued after four weeks because of gastric intolerance. No changes in the Donath- Landsteiner antibody titer could be demonstrated in this period but the clinical course was mild. No Raynaud attacks occurred and the hemolytic ane- mia was less severe than in the previous winter.
MATERIALS AND METHODS
Blood drawn into warm syringes was incubated at 37’~. and the serum separated after two hours. When not tested immediately, the serum was stored at -20’~. Hemolysis tests, including the Donath- Landsteiner test, were perfcrmed as described by Dacie [J3].
Column chromatography of the patient’s serum was performed on DEAE cellulose with stepwise elution by phosphate buffer at pH 6.3, 6.0, 5.8, 5.5 and 4.3, as described by Kim et al. [$I. Guided by the spectrophotometric peaks, the material obtained was pooled in five fractions which were concen- trated over Carbowaxa and finally dialysed against 0.15 hl sodium chloride solution. Elution of the Donath-Landsteiner antibody was performed by the following technic based on that described by Rubin [5]. Normal compatible red cells washed three times with 0.15 M sodium chloride solution were incubated for two hours at 20,. with the pa tient’s serum to which normal guinea pig serum lyophilate had been added to a final dilution of l/10. Subsequently, the red cells were washed five times with 0.15 M sodium chloride solution re- frigerated to 2Oc., the centrifugations being per- formed in a refrigerated centrifuge. Then 0.5 ml. sodium chloride solution, 0.15 M, and a double volume of ether were added to’ the packed red cells. Tire mixture was shaken for two minutes, centri- fuged for ten minutes at 3,000 r.p.m. and finally the supernatant ether was discarded. The eluate, removed from underneath the stroma layer, was
FK. 2. Chromatographic fractions of the patient’s serum. Donath-Landsteiner and Coomhs’ tests were positive with fraction 1 only.
left for thirty minutes at 37Oc. for evaporation ot the last traces of ether.
Analytical ultraccntrifugation was performed in the Weizmann Institute, in a Beckman Model E ultracentrifuge, at 56,200 r.p.m. at 23Oc., protein concentration 0.4 gm. per cent. Immunoelectro- phoresis. micromethod [6] and double diffusion according to Ouchterlony [7], were performed with horse antitotal serum goat anti-IgG serum and rabbit antilambda and antikappa human light chairls.
RESULTS
The Donath-Landsteiner test was positive wit11 both the patient’s red cells and normal 0 Rh positive red cells, with an antibody titer l/8. Complement was necessary both in the c,old and warm phase for the hemolytic reac- tion to take place. With the serum inactivated at .Vi’c., the Donath-Landsteiner test was neg- ative even when complement was added to the test mixture after the cold phase and be- lore incubation at 37”~. Only the addition of complement to the inactivated patient’s serum before refrigeration of the test mixture at 2”~. rendered the Donath-Landsteiner test positive again. Conversely, if after addition of comple- ment and incubation at 2”~. the red cells were washed three times with cold saline solution in a refrigerated centrifuge, no more lysis oc-
VOL. 46, MARCIA 1969
curred when saline solution or inactivated complement was added to the red cells before incubation at 37”~. Hemolysis did occur un- der the conditions described only when normal fresh serum was added to the red cells after washing and before incubation at 37’~. (Table I). This demonstrated that even after fixation of the antibody onto red cells in the presence of complement in the cold phase, additional complement components are necessary in the warm phase for the hemolytic reaction to take place.
FIG. 4. Immunoelectrophoresis with antihuman serum antiserum. a, eluate; b, chromatographic fraction 1.
Frc. 5. Agar double diffusion. a, eluate: b, antihuman IgG antiserum.
FIG. 6. Agar double diffusion. a, eluate; b, urine with lambda light chains (a case of multiple myeloma) ; c, rabbit antilambda light chain antiserum.
test was temperature and complement de- pendent. It was negative when the patient’s serum was incubated at 37”~. with normal compatible erythrocytes. It was strongly posi- tive, up to a dilution of l/ 16 of the patient’s serum in normal serum, when incubation was performed at 2” or 20”~. After inactivation of the patient’s serum the Coombs’ test was neg- ative whatever the incubation temperature, but addition of complement (guinea pig se- rum) rendered it positive again in the tem- perature range indicated.
Donath-Landsteiner tests and indirect Coombs’ tests were performed also with the five fractions obtained by chromatographic fractionation of the patient’s serum. Positive Donath-Landsteiner and Coombs’ tests were obtained at 2” and 20”~. only with fraction I (Fig. 2). Immunoelectrophoretic and ultracen- trifugal analysis showed that fraction 1 was pure IgG globulin of sedimentation constant 6.1 S (Fig. 3 and 4).
The Donath-Landsteiner hemolysin was iso- lated by elution from red cells. By double dif- fusion and immunoelectrophoresis, it could be shown that this hemolysin was a type L IgG globulin, possessing lambda light chains (Fig. 4, 5 and 6).
COMMENTS
The diagnosis of paroxysmal cold hemo- globinuria was established in the present case long after splenectomy. It seems, however, that this was the disease which made the operation necessary. As in Banov’s case [8] splenectomy had a favorable effect which lasted almost ten years. This might be due to a drop in the hemolysin titer following removal of an im- portant mass of antibody-producing tissue.
The treatment of nonsyphilitic cases of par- oxysmal cold hemoglobinuria generally has been unsuccessful. In our case immunosup- pressive treatment was incomplete and of ques- tionable effect.
The Donath-Landsteiner hemolysin has been shown recently to belong to the IgG globu- lins [9]. We could con’firm this finding by immunoelectrophoretic and ultracentrifugal analysis of the chromatographic fractions of the pathologic serum and of the red cell eluate. The present data show that the Donath-Landsteiner hemolysin is in fact an L type IgG globulin, possessing lambda light chains. Cold hemagglutinins generally belong
II) t 1~ Ig11 class, with K type light chains 1 I I ,_ 2 ] .-\lthougli unusual instances of colt1 Ileu~ggiutinins have been reported as being nlixl~d JgG and IgM globulins [I31 or IgM globulins with type L light chains [12]. Con- scclucntly, it appears that neither molecular sile 1101 the type of light chains determines the ~untierythrocytic properties or the low tem- 1)era turr-dependent activity of the antibod)
.I\ already shown by Weiner et al. [IO], and 1Lere confirmed, complement is necessary in the c~)ltl phase for hemolysin fixation on the I-ed cells to take place. But at variance wit11 \\ieiner’s data [IO] our findings imply that be- sides the complement components fixed on the red c:ells in the cold phase, additional complc- men t components are necessary in the warm l)hase for hemolysis to occur. Similarly, Hinz ct al. [I#] have shown that C’l is required in the cold phase and C’2 in the warm phase of the Donath-Landsteiner reaction.
The interpretation of the positive Coombs’ test has been a matter of controversy for some time. Our findings confirm the view that the posi tire Coombs’ test is not related to the presence of an incomplete antibody but with the simultaneous interaction of the erythro- cytes with the Donath-Landsteiner hemolysin and complement.
Lcsvine et al. [15] has shown in four cases of paroxysmal cold hemoglobinuria that the Do- nath-Landsteiner antibody has anti P-P,(Tja) specificity. Thus it appears that, like the warm antibodies [16] and the cold agglutinin [17], the Donath-Landsteiner hemolysins also have distinct blood group specificity.
Ac,KnowEedgment: The technical collabora- tion of N. Ben-Joseph and S. Shapiro is gratefully acknowledged. We express our thanks to Dr. A. Gottlieb, Head of the De- partment of Pediatrics, Zahalon Hospital, for referring the patient to us; Prof. D. Brunner, Head of the Department of Medicine, where the patient was hospitalized; Dr. A. Frensdorf, Dr. 1. Schwartz, Dr. A. Pinto and Mr. E. Ro-
senfcltl, lrom the Departrnc~r~c 01 Imnl~~nology, Tel-:\\ i\ IYniversity, for their bell) with f’ncili- tie5 ;11lt1 rcagmts.
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